This invention relates to molecules with endonucleolytic activity and enhanced half-lives useful in the site-specific cleavage of RNA. This invention also relates to the control of gene expression through the degradation of mRNA.
Ribozymes are RNA molecules with catalytic activities including the ability to cleave at specific phosphodiester linkages in RNA molecules to which they have hybridized, such as mRNAs, RNA-containing substrates, and ribozymes themselves.
Ribozymes may assume one of several physical structures, one of which is called a "hammerhead" (Haseloff and Gerlach (1988) Nature 334:585-591). A hammerhead ribozyme is composed of a catalytic core containing nine conserved bases, a double-stranded stem and loop structure (helix II), and two regions flanking the catalytic core that are complementary to the target RNA. The flanking regions enable the ribozyme to bind to the target RNA specifically by forming double-stranded stems I and III. Cleavage occurs in cis (i.e., cleavage of the same RNA molecule that contains the hammerhead motif) or in trans (cleavage of an RNA substrate other than that containing the ribozyme) next to specific ribonucleotide triplet by a transesterification reaction from a 3',5'-phosphate diester to a 2',3'-cyclic phosphate diester. It is currently believed that this catalytic activity requires the presence of specific, highly conserved sequences in the catalytic region of the ribozyme.
Although the endonucleolytic activity of ribozymes has been demonstrated in vitro, their use in vivo has been limited by their susceptibility to RNAses. Furthermore, therapeutics such as ribozymes having greater than 30 or more nucleotides are expensive and difficult to produce in great quantities. Thus, there is a need for smaller molecules with increased nuclease resistance that can be used to cleave RNA in vitro and in vivo, and to control gene expression for in vitro studies and in vivo therapeutic use.
In an effort to protect antisense oligonucleotides from degradative influences in vivo, various structural modifications have been made to these molecules, including the replacement of phosphodiester linkages with non-phosphodiester linkages, the substitution of various sugar groups and bases, the addition of end-capping groups, and the substitution or replacement of existing structures with the self-hybridizing termini (reviewed in Goodchild (1990) Bioconjugate Chem. 1:165-187; Agrawal et al. (1992) Trend Biotechnol. 10:152-158; WO 93/15194; WO 94/10301; WO 94/12633).
However, modifications which protect an RNA molecule from endonuclease digestion may also affect the catalytic activity of the ribozyme. For example, Perreault et al. (Nature (1990) 344:565-567) report that the replacement of ribonucleotides at various conserved positions within the ribozyme sequence with 2'-deoxynucleotides resulted in a 96% decrease of catalytic efficiency. Perreault et al. (Biochem. (1991) 30:4020-4025) and Dahm et al. (Biochim. (1990) 72:819-23) disclose that the replacement of various 2'-hydroxyl groups with hydrogen atoms reduced the catalytic activity of hammerhead ribozymes. Olsen et al. (Biochem. (1991) 30:9735 -9741) report that replacing 2'-hydroxyl groups on all adenosine residues by either fluorine or hydrogen decreases the catalytic activity of a ribozyme. Odai et al. (FEBS Lett. (1990) 267:150 -152) report that replacing the exocyclic amino group of a conserved guanosine residue in the core region with hydrogen reduced catalytic activity. Ruffner et al. (Nucleic Acids Res. (1990) 18:6025-6029) and Buzayan et al. (Nucleic. Acids Res. (1990) 18:4447-4451) disclose that replacing oxygen atoms by sulfur on various internucleotide phosphate residues reduces catalytic activity. Pieken et al. (Science (1991) 253:314-317) disclose that catalytic activity is reduced when various 2'-hydroxyl groups on adenosine residues are replaced with fluorine and when the 2'-hydroxyl groups on cytidine residues are replaced with amine groups. Paolella et al. (EMBO J. (1992) 11:1913-1919) have investigated which 2'-hydroxyl groups may or may not be alkylated without loss of catalytic activity.
Other groups have substituted nucleotides within the ribozyme with nucleotide analogs. For example, Usman et al. (WO 93/15187) designed chimeric polymers or "nucleozymes" with ribozyme-like catalytic activity having ribonucleotides or nucleic acid analogs (with modified sugar, phosphate, or base) at catalytically critical sites and nucleicacid analogs or deoxyribonucleotides at non-catalytically critical sites.
Recently, modifications such as a reduction in the length of the helix II structure of the hammerhead ribozyme have been made in an effort to design a more stable molecule without reducing its catalytic activity (see, e.g., Goodchild et al. (1991) Arch. Biochem. Biophys. 284:386-391; Tuschl et al. (1993) Proc. Nail. Acad. Sci. (USA) 90:6991-6994; McCall et al. (1992) Proc. Natl. Acad. Sci. (USA) 89:5710-5714).
Modifications in ribozyme structure have also included the substitution or replacement of various non-core portions of the molecule with non-nucleotidic molecules (see, e.g., Benseler et al. (1993) J. Am. Chem. Soc. 115:8483-8484; Ma et al. (1993) Biochem. 32:1751-1758; Ma et al. (1993) Nucleic Acids Res. 21:2585-2589; and Thomson et al. (1993) Nucleic Acids Res. 21:5600-5603).
Thus, ribozymes with improved resistance to endonucleolytic digestion are desirable, and there continues to be a need for such catalytic molecules.